The following is an outline of the procedures for testing mycoplasma contamination in tissue culture cells. As a rule, anytime you freeze down cells, one plate of cells should be processed for testing. In addition, we will routinely test the cell lines that are the backbone of our research (e.g. Mcf-7 WT and CMV) approximately every three months. The test involves amplification of a small segment of the 16S rRNA from the mycoplasma genome.
All of the reagents necessary to perform this test can be found in Freezer 4 and on the tissue culture shelf.
Preparing cells for testing:
Harvest one plate (100mm) of cells (~60-70% confluent). [Treat with Trypsin then add 10 mLs of media and spin in the clinical centrifuge for 5-10 minutes (set at max). Or simply harvest the cells with a rubber policeman in their media and pellet in the clinical for 5 minutes at maximum speed.]
Wash this cell pellet twice with PBS and store the pellet in an eppendorf tube in Freezer 4 (2nd shelf from the top) in the mycoplasma testing box.
Making cell lysates:
Add 100 µL of cell lysis buffer (50 mM Kcl, 10 mM Tris-Hcl pH 8.3, 2.5 mM MgCl2, 0.1 mg/mL gelatin, 0.45% NP-40 and 0.45% Tween-20). Add the proteinase K to 6 µg and incubate at 60°C for 1 hour.
Transfer tubes to 90°C for 10 minutes. Quick spin in the microcentrifuge to remove debris.
Make a master mix of dNTP, PCR buffer, primers and water.
Each reaction should contain the following:
1 µL of 5 µM Forward Primer
1 µL of 5 µM Backward Primer
0.5 µL of 5 mM dNTP
1.5 µL of 10X PCR buffer (1.5 mM MgCl2, 50 mM Kcl, 10mM Tris-HCl pH 8.3, and 0.0001% gelatin)
10 µL of H2O
1 µL of lysate
The PCR requires a hot start -94°C for 5 minutes.
The program for the PCR is stored under "myco". There are two programs depending on the primers chosen. Outer-for outer primers and Inner-for the inner primers.
After the 5 minute incubation at 94°C add 10 µL of Taq Buffer (8 µL of H2O, 1 µL of 10 X PCR buffer and 1 µL of Taq (1 Unit)).
Outer program: 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 minute - for 30 cycles. Final extension at 72°C for 10 minutes.
Visualize your results on a 1-2% TAE Agarose gel. (Load 10 µL of the PCR reaction).